This Phase II SBIR proposes to develop a microfluidic cell culture and analysis platform for the in vitro screening of cancer cells. The end result of this research will be to deliver a fully functional system (automated instrument and disposable microfluidics) that can be used in both research and pharmaceutical labs. This will be validated and applied to the research underway at our collaborating institute for the purpose of profiling the Raf-MEK-ERK pathway in a panel of ~60 breast cancer cell lines for improved prediction of therapeutic response. [unreadable] [unreadable] The microfluidic platform will provide key advantages over the current cell based screening technology (96-well plate based), including: 1) improved handling of small cell samples (micoliters per array), 2) the ability to design more relevant microenvironments for phenotype analysis, 3) enabling multiplexed continuous flow experimentation, and 4) 10X-100X reduction of time and cost for cell culture automation. In addition, the platform is designed such that application specific microfluidic arrays can be utilized with a single system, increasing the flexibility and impact of the technology. [unreadable] [unreadable] The first major aim of this project will be to engineer an automated microfluidic screening platform. The main tasks are to optimize the design of the Phase I prototype, scale-up to a 384 well format, and refine the control system for high throughput operation. Three key innovations developed in our previous work will be further expanded to complete this aim: 1) the design of microfluidic networks and perfusion barriers to better approximate in vivo culture conditions, 2) the use of a pneumatic pressure driven manifold for multiplexed, non-wetted pumping of nanoliter volumes, and 3) the fabrication process that enables formatting the microfluidic arrays to SBS standards, making it compatible with current 96 and 384 well robotic screening instrumentation. [unreadable] [unreadable] The second major aim will be to apply this system to the cancer cell screening program at our collaborating institute. This will address areas where microfluidic technology can offer enabling benefits not possible with existing tools. Specific applications include: 1) flow based drug exposure, 2) cell invasion assay, 3) 3D ECM culture, 4) medium conditioning by stromal cells, and 5) integration with RNAi methods. [unreadable] [unreadable] [unreadable]